Methods and compositions using butyrate esters of threitol

ABSTRACT

The present invention relates to the use of butyrate esters of threitol, alone or in combination with other agents, in pharmaceutical compositions and methods for increasing fetal hemoglobin and gamma globin in a patient. These methods are particularly useful in treating β-hemoglobinopathies, such as sickle cell syndromes and β-thalassemia syndromes. Compositions comprising butyrate esters of threitol, alone or in combination antiproliferative and differentiating agents are also useful in methods for inducing cell differentiation in malignant cells. These methods are useful in treating cancer, particularly the tetrabutyrate ester.

This is a division of application Ser. No. 08/550,453, filed Oct. 30,1995, now U.S. Pat. No. 5,763,488, entitled METHODS AND COMPOSITIONSUSING BUTYRATE ESTERS OF THREITOL.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the use of butyrate esters of threitol,alone or in combination with other agents, in pharmaceuticalcompositions and methods for increasing fetal hemoglobin and gammaglobin in a patient. These methods are particularly useful in treatingβ-hemoglobinopathies, such as sickle cell syndromes and β-thalassemiasyndromes. Compositions comprising butyrate esters of threitol, alone orin combination antiproliferative and differentiating agents are alsouseful in methods for inducing cell differentiation in malignant cells.These methods are useful in treating cancer, particularly thetetrabutyrate ester.

BACKGROUND OF THE INVENTION

β-hemoglobinopathies are a group of inherited disorders of β-globinbiosynthesis. Although efforts have concentrated on a variety oftherapeutic regimens, feasible clinical treatments for thesedebilitating diseases remain scarce.

Various therapies have been utilized in the treatment ofβ-hemoglobinopathies, each accompanied by drawbacks G. P. Rogers et al.,"Current and Future Strategies for the Managements of Hemoglobinopathiesand Thalassemia", Hematology 1994, Education Program American Society ofHematology, pp. 9-20 (1994)!. Although hydroxyurea stimulates fetalhemoglobin production and reduces sickling crisis in sickle cell anemiapatients, its use is potentially limited by myelotoxicity and the riskof carcinogenesis. Potential long term carcinogenicity is also adrawback of 5-azacytidine-based therapies. Red blood cell transfusionsexpose patients to the potential of a wide range of infectious viralagents, alloimmunization and iron overload. Bone marrow transplants arenot a readily available option for a large number of patients.Erythropoietin-based therapies have not proved consistent among a rangeof patient populations. Such varying drawbacks contraindicate the longterm use of such agents or therapies.

It is clear from multicenter studies involving numerous patients withsickle cell disease that increased blood levels of fetal hemoglobin areassociated with lower events of sickle cell crisis and longer survivaltime Platt et al., "Pain in Sickle Cell Disease, New Eng. J. Med., 325,pp. 11-16 (1991); Platt et al., "Mortality ion Sickle Cell Disease", NewEng. J. Med., 330, pp. 1639-44 (1994)!. Accordingly, in an effort toavoid the disadvantages of conventional therapies forβ-hemoglobinopathies, therapies have centered around ways to increasefetal hemoglobin production. Recent clinical trials have focused on theuse of butyrate analogs, including arginine butyrate and isobutyramide,to stimulate fetal hemoglobin production as a means of treatment S.Perrine et al., "A Short Term Trial of Butyrate to StimulateFetal-Globin-Gene Expression in the β-globin Disorders", N. Eng. J.Med., 328, pp. 81-86 (1993); S. P. Perrine et al., "Isobutyramide, anOrally Bioavailable Butyrate Analogue, Stimulates Fetal Globin GeneExpression in vitro and in vivo," British J. Haematology, 88, pp. 555-61(1994); A. F. Collins et al., "Oral Sodium Phenylbutyrate Therapy inHomozygous β Thalassemia: A Clinical Trial", Blood, 85, pp. 43-49(1995)!. Clinical trials have also employed sodium phenylbutyrate as ahemoglobin switching agent for β-thalassemia Collins et al., supra!.

Following the observation that butyric acid induces cell differentiationin vitro A. Leder and P. Leder, "Butyric Acid, a Potent Inducer ofErythroid Differentiation in Cultured Erythroleukemic Cells", Cell, 5,pp. 319-22 (1975)!, that compound was found to demonstrate promisingeffects in leukemia patients, by inducing cell differentiation A.Novogrodsky et al., "Effect of Polar Organic Compounds on LeukemicCells", Cancer, 51, pp. 9-14 (1983)!. Aside from their use in treatingβ-hemoglobinopathies, butyrate derivatives such as arginine butyrate, anarginine salt of butyric acid, have been shown to exert anti-tumor andanti-leukemia effects in mice C. Chany and I. Cerutti, "Antitumor EffectOf Arginine Butyrate in Conjunction with Corynebacterium Parvum andInterferon", Int. J. Cancer, 30, pp. 489-93 (1982); M. Otaka et al.,"Antibody-Mediated Targeting of Differentiation Inducers To Tumor Cells:Inhibition of Colonic Cancer Cell Growth in vitro and in vivo", Biochem.Biophys. Res. Commun., 158, pp. 202-08 (1989)!.

Although butyrate salts have the advantage of low toxicity as comparedwith conventional chemotherapeutic agents, their short half-lives invivo have been viewed as a potential obstacle in clinical settings A.Miller et al., "Clinical Pharmacology of Sodium Butyrate in Patientswith Acute Leukemia", Eur. J. Clin. Oncol., 23, pp. 1283-87 (1987);Novogrodsky et al., supra!. The rapid clearance of these agents resultsin an inability to deliver and maintain high plasma levels of butyrateand necessitates administration by intravenous infusion. Anotherpotential obstacle to the use of butyrate salts is salt overload and itsphysiological sequelae.

In view of these observations, various prodrugs of butyric acid havebeen proposed for use in β-hemoglobinopathy and leukemia differentiationtherapies. Such prodrugs include tributyrin and n-butyric acid mono- andpolyesters derived from monosaccharides Z. Chen and T. Breitman,"Tributyrin: A Prodrug of Butyric Acid for Potential ClinicalApplication in Differentiation Therapy", Cancer Res., 54, pp. 3494-99(1994); H. Newmark et al., "Butyrate as a Differentiating Agent:Pharmacokinetics, Analogues and Current Status", Cancer Letts., 78, pp.1-5 (1994); P. Pouillart et al., "Pharmacokinetic Studies of N-ButyricAcid Mono- and Polyesters Derived From Monosaccharides", J. Pharm. Sci.,81, pp. 241-44 (1992)!. Such prodrugs have not proved useful astherapeutics, however, due to factors such as low bioavailability, lackof effective oral deliverability, short half life, low C_(max) or highpharmacokinetic variability. Other prodrugs, such as AN-9 and AN-10,elicit metabolites that may produce formaldehyde in vivo, which may leadto toxic effects in patients.

To date, conventional methods and therapeutic agents have not proved tobe safe and effective for all patients in the long term treatment ofβ-hemoglobinopathies. This is also the case for diseases characterizedby neoplastic, tumorigenic or malignant cell growth, or malignanthematological disorders. Accordingly, the need exists for alternativeshaving advantages over, and avoiding the disadvantages of, suchconventional methods and agents, while providing effective therapy forthose target diseases.

DISCLOSURE OF THE INVENTION

The present invention solves these problems by providing methods andcompositions utilizing butyrate esters of threitol for increasing fetalhemoglobin and gamma globin production in a patient. These butyrateesters demonstrate good bioavailability, effective oral deliverability,good half-life, good C_(max) and surprisingly low pharmacokineticvariability between individual patients. This last factor increasestheir utility as agents to deliver therapeutically effective amounts ofsystemic butyrate.

The compositions and methods of the invention are especially useful fortreating or reducing the advancement, severity, symptoms or effects ofβ-hemoglobinopathies, including sickle cell syndromes and β-thalassemiasyndromes. In addition, the methods and compositions according to thepresent invention are useful for stimulating cell differentiation inmalignant cells. Such compositions and methods are useful for treatingcancer.

Accordingly, the methods and compositions of this invention are notbeset by the variety of side effects which typically characterizeconventional therapy regimens.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth.

According to one embodiment, this invention provides pharmaceuticalcompositions comprising a butyrate ester of threitol represented by theformula: ##STR1## wherein R is ##STR2## or hydrogen, provided that atleast one R is ##STR3## and the stereochemistry at the achiral carbonsis independently selected from R or S and a pharmaceutically acceptablecarrier or adjuvant. Preferably each R is ##STR4## More preferably, thepharmaceutical compositions of this invention comprise an approximatelyequimolar mixture of (R) and (S) configurations presented by thefollowing Fischer projections: ##STR5##

The tetrabutyrate esters of threitol and the partially esterifiedanalogs of threitol useful in the methods and compositions of thepresent invention may be synthesized by conventional techniques.Advantageously, these compounds are conveniently synthesized fromcommercially available starting materials. For example, they may beprepared from the D-, L- or D,L-forms of threitol using an appropriateactivated reagent, such as an activated butyric acid derivative, inconventional esterification techniques. For instance, reaction with anactivated carboxylate, such as an acyl halide (e.g., acid fluorides,acid chlorides, and acid bromides), an acyl cyamide of acyl imidazolide,an activated ester such as nitrophenyl ester or 1-hydroxysuccinimide(HOSu) ester, an anhydride such as the symmetrical anhydride or isobutylmixed carbonic anhydride, or mixed carbonic-phosphoric orcarbonic-phosphonic anhydrides with an appropriate alcohol, will yieldthe corresponding ester.

Other methods of forming esters from alcohols and carboxylic acids ortheir derivatives are also well known to those of skill in the art.These include removal of water by Dean-Stark distillation, Fischeresterification and transesterification. Various catalysts and additives,including protic and/or Lewis acids, bases or zeolites may be used toincrease the ease or efficiency of these reactions. Enzymatic methods toform esters are also well known in the art.

Specific modifications of these methods, as well as other means offorming esters are known by those of skill in the art. It will bereadily recognized that in order to facilitate specific reactions, theprotection of one or more potentially reactive groups followed bysubsequent removal of that group may be required. Such modifications arewithin the skill of the art.

In any synthesis method, the desired compound may be isolated by anytechnique, including, for example, distillation, chromatographictechniques, such as normal phase, reverse phase, ion-exchange, affinity,or gel permeation, as well as extraction, crystallization, or othermeans. The relative ease of synthesis of the compounds of this inventionrepresents an advantage in their large scale production.

It should also be understood that the butyrate esters of threitol usedin the compositions of this invention may be modified by appendingappropriate functionalities to enhance selective biological properties.Such modifications are well recognized in the art and include sustenanceof plasma and/or whole blood butyrate concentration, increased oralavailability, altered metabolism and altered rate of excretion ofbutyric acid or butyric acid prodrugs.

The pharmaceutical compositions of this invention are characterized bythe presence of a butyrate ester of threitol in an amount effective toincrease the production of fetal hemoglobin or stimulate celldifferentiation in a patient and a pharmaceutically acceptable carrieror adjuvant. More specifically, these compositions are designed to treata patient suffering from a β-hemoglobinopathy or a malignant disease.The term "malignant disease", as used herein denotes a conditioncharacterized by neoplastic, tumorigenic or malignant cell growth, or ahematological disorder.

An amount effective to increase the production of fetal hemoglobin orstimulate cell differentiation in a patient will depend, of course, onthe particular disease to be treated, the severity of the disease, thephysical condition of the patient and the judgment of the treatingphysician. Preferably, the prodrug of Formula I will be present in anamount capable of producing a plasma butyric acid concentration ofbetween about 0.03 mM and 3.0 mM within 8 hours of administration. Morepreferably, the prodrug of Formula I is present in an amount thatproduces a plasma butyric acid concentration of between about 0.1 mM and1.0 mM within 6 hours of administration. Most preferably, the prodrug inthe composition is present in an amount that produces a plasma butyricacid concentration of between about 0.1 mM and 1.0 mM within 2 hours ofadministration and the concentration remains at those levels for atleast 2 hours. Dosages of between 25 mg/kg and 3000 mg/kg body weight ofbutyrate ester of threitol administered one or more time per day willproduce the desired serum butyrate concentration. Preferably, thepatient will be administered the prodrug between 1 and 4 times per day.

In a preferred embodiment, these compositions additionally comprise aconventional agent used in the treatment of β-hemoglobinopathies. Theconventional agent may be present in the same amount or less than thatnormally required to treat β-hemoglobinopathies in a monotherapy. Thenormal dosages of these conventional agents are well known in the art.Such agents include hydroxyurea, clotrimazole, isobutyramide,erythropoietin and salts of short-chain fatty acids, such asphenylacetic acid, phenylbutyric acid and valproic acid.

According to an alternate preferred embodiment, the compositionscomprise a butyrate ester of threitol and a conventional agent used inthe treatment of diseases characterized by neoplastic, tumorigenic ormalignant cell growth, or a hematological disorder in a patient. Thisadditional agent may be present in an amount equal to or less than thatnormally required to treat such diseases in a monotherapy. The normaldosages of these conventional agents are well known in the art. Suchagents include, erythropoietin, or cancer chemotherapeutic agents, suchas hydroxyurea or 5-azacytidine.

The carriers and adjuvants useful in the pharmaceutical compositions ofthis invention include, for example, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances, such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes such as protamine sulfate, disodium hydrogenphosphate, sodium chloride, zinc salts, colloidal silica, magnesium,trisilicate, polyvinyl pyrrolidone, cellulose-based substances andpolyethylene glycol. Adjuvants for topical or gel base forms may beselected from the group consisting of sodium carboxymethylcellulose,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers,polyethylene glycol and wood wax alcohols.

The compositions of the present invention may be in a variety ofconventional depot forms. These include, for example, solid, semi-solidand liquid dosage forms, such as tablets, pills, powders, liquidsolutions or suspensions, oil dilutions, liposomes, capsules,suppositories, injectable and infusible solutions. The preferred formdepends upon the intended mode of administration and therapeuticapplication.

For example, oral administration may be by any orally acceptable dosageform including, but not limited to, capsules, tablets, and aqueous ornon-aqueous suspensions and solutions. In the case of tablets for oraluse, carriers which are commonly used include lactose and corn starch.Lubricating agents, such as magnesium stearate, are also typicallyadded. For oral administration in a hard gelatin capsule form, usefuldiluents include lactose and dried corn starch. Soft gelatin capsulesincorporating oils and/or polyethylene glycols as excipients may also beused. Fluid unit dosage forms for oral administration include syrups andsuspensions. For example, the butyrate ester of threitol may bedissolved in an aqueous vehicle together with sugar, sweetening orflavoring agents and preservatives to form a syrup. Suspensions may beprepared with an aqueous vehicle and a dispersing agent, such as acacia,tragacanth or methylcellulose.

Preferably, the pharmaceutical compositions of this invention areformulated for oral administration. Even more preferred are oralemulsions comprising between about 5 to 40% (w/w) of the prodrug offormula I and an ionic or non-ionic surfactant with the resultingcomposition having an HLB value of between 0-40. Preferred surfactantsinclude Tween-20, Tween-80, Spam-20, Spam-40 and poloxamers, such asS-108.

The butyrate esters of threitol which characterize the compositions ofthis invention are characterized by several advantages. They aremetabolized to yield therapeutic butyric acid plasma concentrations overa sustained period of time, resulting in therapeutically effectiveexposure to butyric acid. Additionally, they are orally bioavailable,unlike sodium butyrate--which is rapidly cleared before therapeuticplasma concentration levels can be reached. They are also non-toxic,thus avoiding, for example, sodium overload and irritation which may beassociated with injections of hyperosmolalic solutions.

The most surprising and unexpected feature of the butyrate esters ofthreitol that characterize the pharmaceutical compositions of thisinvention is that they exhibit significantly lower pharmacokineticvariability over conventional prodrugs of butyric acid. Pharmacokineticvariability is the measure of differences in the serum butyrateconcentrations between different patients administered the same amountof butyrate esters of threitol. Pharmacokinetic variability isquantified by dividing the standard deviation for a given parameter,such as C_(max) or AUC, by the mean value of that parameter, in a seriesof patients given dose the same dosage of butyrate ester of threitol.

In particular, the tetrabutyrate ester of mesothreitol is characterizedby a pharmacokinetic variability of 35-40%, as compared withconventional prodrugs of butyric acid, such as tributyrin, which has apharmacokinetic variability of 70%. This reduced variability means thatthe compositions of this invention provide sustained release and producemore consistent plasma concentrations of butyric acid among individualpatients. This, in turn, minimizes the potential for cellular toxicity,which, based on our own in vitro cell culture studies, has been observedat butyric acid concentrations above 3.0 mM under certain in vitroconditions.

According to another embodiment, the invention provides methods fortreating a β-hemoglobinopathy in a patient. This method comprises thestep of treating the patient with any of the compositions describedabove. The term "treating", as used herein includes reducing theseverity, symptoms or effects of the β-hemoglobinopathy.

Preferably, the method provides a serum butyric acid concentration ofbetween about 0.03 mM and 3.0 mM within about 8 hours of administration.More preferably, this produces a plasma butyric acid concentration ofbetween about 0.1 mM and 1.0 mM within about 6 hours of administration.Most preferably, the prodrug in the composition is present in an amountthat produces a plasma butyric acid concentration of between about 0.1mM and 1.0 mM within 2 hours of administration and the concentrationremains within that range for at least 2 hours. These plasma levels areachieved by administering a butyrate ester of threitol to the patient ata dose of between about 25-3000 mg/kg body weight one or more times perday. Preferably, the patient will be administered the prodrug between 1and 4 times per day.

The β-hemoglobinopathies which may be treated by this method includesickle cell syndromes, such as sickle cell anemia, hemoglobin SCdisease, hemoglobin SS disease and sickle β-thalassemia; β-thalassemiasyndromes, such as β-thalassemia; other genetic mutations of theβ-globin gene locus that lead to unstable hemoglobins, such ascongenital Heinz body anemia, β-globin mutants with abnormal oxygenaffinity and structural mutants of β-globin that result in thalassemicphenotype. These diseases are described in The Molecular Basis of BloodDisease, vol. II, G. Stamatoyannopoulos et at., eds., pp. 157-244(1994).

According to a preferred embodiment, the above-described methodcomprises the additional step of treating the patient with an agent thatis normally used to treat such β-hemoglobinopathies. That agent may beadministered prior to, sequentially with or after treatment with thebutyrate prodrug-containing composition. Of course, if the compositionused to treat the disease is one that already contains such conventionalagent, this additional step can be omitted.

The amount of conventional agent administered in these methods ispreferably less than that normally required to treat such diseases in amonotherapy. The normal dosages of these conventional agents are wellknown in the art. Such agents include hydroxyurea, clotrimazole,isobutyramide, erythropoietin and salts of short-chain fatty acids, suchas phenylacetic acid, phenylbutyric acid and valproic acid.

According to another embodiment, the invention provides method fortreating diseases characterized by neoplastic, tumorigenic or malignantcell growth, as well as malignant hematological disorders. Treatmentincludes prevention of the progression of the disease or its recurrence.Such diseases include carcinomas, myelomas, melanomas, lymphomas andleukemias. Preferably, the method provides the same serum butyric acidconcentrations indicated above as being desirable for treatingβ-hemoglobinopathies.

According to a preferred embodiment, the above-described methodcomprises the additional step of treating the patient with an agent thatis normally used to treat such malignancies. That agent may beadministered prior to, sequentially with or after treatment with thebutyrate prodrug-containing composition. Of course, if the compositionused to treat the disease is one that already contains such conventionalagent, this additional step can be omitted.

The amount of conventional agent administered in these methods ispreferably less than that normally required to treat such diseases in amonotherapy. In those instances, the occurrence of any side effectsassociated with that agent may be reduced or avoided. The normal dosagesof these conventional agents are well known in the art. Such agentsinclude, erythropoietin, or cancer chemotherapeutic agents, such ashydroxyurea or 5-azacytidine.

Combination therapies with conventional agents according to thisinvention (whether part of a single composition or administered separatefrom the prodrugs of this invention) may also exert an additive orsynergistic effect, particularly when each component acts to treat orprevent the target disease via a different mechanism.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are set forth for illustrative purposesonly and are not to be construed as limiting this invention in anymanner.

EXAMPLE 1 Synthesis of d-, l- and d,l-Threitol Tetrabutyrate Esters

We synthesized d-threitol tetrabutyrate ester as follows. We added 17 mlof Et₃ N to 3.0 g of D-threitol dissolved in CH₂ Cl₂ and cooled themixture to 0° C. We then added 11 ml of CH₃ (CH₂)₂ COCl in 5 ml CH₂ Cl₂over 30 minutes. The reaction mixture was stirred at room temperatureovernight. We then diluted the reaction with ether and filtered theresulting material. The precipitate was washed once over a filter withether. The filtrates were combined, washed twice with water, once withsaturated NaCl and dried over MgSO₄, filtered and concentrated.

The resulting yellow oil was run on an MPLC column in 90:10(hexane:EtoAc). The yield of purified product was 8.35g of a lightyellow oil.

We synthesized d-, l-threitol tetrabutyrate ester using the samemethodology replacing D-threitol with D,L-threitol using the same ratioof equivalents (1:5:4.5; threitol:Et₃ N:CH₃ (CH₂)₂ COCl).

The structure of the purified compounds was confirmed by NMR.

EXAMPLE 2 Oral Availability of Butyrate Esters of Threitol in Rats

We evaluated oral bioavailability and sustenance of plasmaconcentrations of butyric acid in rats receiving tetrabutyrate esters ofthreitol and tributyrin by oral gavage.

This assay was carried out according to the protocol described in Danielet al., Clinca Chimica Acta., 181, pp. 255-64 (1989); Planchon et al.,J. Pharm. Sci., 82, pp. 1046-48 (1993); Pouillart et al., J. Pharm Sci.,81, pp. 241-44 (1992). Each compound was tested in five to six rats(Sprague Dawley; Harlan Labs, Inc.) weighing approximately 300 gramseach. The relevant pharmacokinetic parameters for these agents arelisted in the table below. In that table, data are expressed as mean ±standard deviation (range) and were compared using the unpairedStudent's t-test.

                                      TABLE 1.                                    __________________________________________________________________________    Pharmokinetics of butyrate esters of threitol in rats.                                  Dose                                                                              Number of                                                                          AUC   C.sub.max                                                                           Plasma t.sub.1/2                               Compound  (gm/kg)                                                                           animals                                                                            (mM · hr)                                                                  (mM)  (mins)                                         __________________________________________________________________________    tributyrin                                                                              3.0 6    1.59 ± 0.93                                                                      0.51 ± 0.46                                                                      157.2 ± 70.2                                l-threitol tetrabutyrate                                                                2.4 5    1.20 ± 0.40                                                                      0.58 ± 0.13                                                                       66.0 ± 12.0                                ester                                                                         d-threitol                                                                              2.8 6    0.90 ± 0.26                                                                      0.47 ± 0.14                                                                       58.8 ± 19.8                                tetrabutyrate ester                                                           dl-threitol                                                                             3.0 5    1.25 ± 0.22                                                                      0.39 ± 0.12                                                                      106.2 ± 22.2*                               tetrabutyrate ester                                                           __________________________________________________________________________     * Significantly different from lthreitol tetrabutyrate ester and dthreito     tetrabutyrate ester (p < 0.01).                                          

As shown above, the tetrabutyrate esters of threitol were found torelease butyric acid in vivo with lower variability than tributyrin.Oral administration of each of those prodrugs of butyric acid alsoincreased the "apparent" plasma half-life of butyric acid tosignificantly longer than the 6 minutes observed in leukemia patientsafter continuous infusions of sodium butyrate Miller et al., supra!. Thearea under the plasma concentration-time curve and the observed C_(max)values for butyric acid were not significantly different for any of thetetrabutyrate esters or tributyrin (following dose-normalization).Advantageously and unexpectedly, the butyrate ester of D,L-threitol wasfound to have an "apparent" butyric acid plasma half-life which wassignificantly longer than that observed for the tetrabutyrate ester ofeither D-threitol or L-threitol.

A closer examination of the data showed that the release of butyric acidfrom tributyrin was more variable when compared to that obtainedfollowing oral administration of the tetrabutyrate ester ofD,L-threitol. The pharmacokinetic variability with tributyrin oraladministration is demonstrated by the wide range of C_(max) (0.295 to1.46 mM); AUC (0.72 to 3.23 mM.hr); and plasma half-life (67.2 to 259.8minutes). On the other hand, the ranges for C_(max), AUC and plasmahalf-life for orally administered tetrabutyrate ester of D,L-threitolwere 0.26 to 0.545 mM, 0.95 to 1.53 mM.hr and 79.2 to 126.9 minutes,respectively. Thus, both tributyrin and the tetrabutyrate ester ofD,L-threitol are orally bioavailable to provide therapeutic plasmalevels of butyric acid. However, a clear advantage of that tetrabutyrateester is its reduced pharmacokinetic variability--signifying aconsistent pharmacokinetic profile which, in turn, enables more reliableclinical treatment regimens than those based on tributyrin.

While we have hereinbefore described a number of embodiments of thisinvention, it is apparent that our basic constructions can be altered toprovide other embodiments which utilize the processes and compositionsof this invention. Therefore, it will be appreciated that the scope ofthis invention is to be defined by the claims appended hereto ratherthan by the specific embodiments which have been presented hereinbeforeby way of example.

We claim:
 1. A method of inducing differentiation in a malignant cellcomprising the step of contacting said cell with a pharmaceuticalcomposition comprising:a. an amount of a butyrate ester of threitoleffective to induce differentiation in malignant cells sensitive to saidbutyrate ester in a patient; and b. a pharmaceutically acceptableadjuvant or carrier;said butyrate ester of threitol having the formula:##STR6## wherein each R is independently selected from ##STR7## .
 2. Themethod according to claim 1, wherein said pharmaceutical compositioncomprises a mixture of ##STR8## .
 3. The method according to any one ofclaims 1, or 2, wherein said pharmaceutical composition is an emulsionformulated for oral administration.
 4. The method according to any oneof claims 1, or 3, wherein said method is used to treat cancer in apatient.
 5. The method according to claim 4, wherein said methodproduces a serum butyric acid concentration of between about 0.1 mM and1.0 mM within 2 hours of administration and the serum butyric acidconcentration remains between about 0.1 mM and 1.0 mM for at least 2hours.
 6. The method according to claim 4, wherein said cancer isselected from the group consisting of carcinomas, myelomas, melanomas,lymphomas and leukemias.
 7. The method according to claim 4, whereinsaid pharmaceutical composition is administered to said patient orally.8. The method according to claim 4, comprising the additional step ofadministering to said patient an agent used in the treatment of diseasescharacterized by neoplastic, tumorigenic or malignant cell growth in anamount equal to or less than an amount of said agent used in amonotherapy.
 9. The method according to claim 8, wherein said agent iserythropoietin.
 10. The method according to claim 8, wherein said agentis hydroxyurea or 5-azacytidine.
 11. A pharmaceutical compositioncomprising:a. an amount of a butyrate ester of threitol effective toinduce differentiation in malignant cells sensitive to said butyrateester in a patient; b. an amount of an agent used in the treatment ofdiseases characterized by neoplastic, tumorigenic or malignant cellgrowth equal to or less than an amount of said agent used in amonotherapy; and c. a pharmaceutically acceptable adjuvant or carrier;said butyrate ester of threitol having the formula: ##STR9## whereineach R is independently selected from ##STR10## .
 12. The pharmaceuticalcomposition according to claim 11, wherein said agent is erythropoietin.13. The pharmaceutical composition according to claim 11, wherein saidagent is hydroxyurea or 5-azacytidine.
 14. The pharmaceuticalcomposition according to claim 7, comprising a mixture of ##STR11## .15. The pharmaceutical composition according to any one of claims 14,11, 12 or 13, wherein said composition is an emulsion formulated fororal administration.